NSWHP - PR - 062 - Antibody Screening and Identification Test Methods

Antibody Screening and Identification Test
Methods
NSWHP_PR_062
1. Purpose
This document outlines the method for performing antibody screening and identification tests for
transfusion samples across NSW Health Pathology Laboratories. The aim of antibody screening is to
detect atypical antibodies which may be present in the patient's plasma or serum.
1.1. Principle of Assay

The routine method for alloantibody detection and identification is an indirect antiglobulin test (IAT)
performed using Bio-Rad column agglutination technology (CAT). Polyspecific anti human globulin
(AHG) reagents containing anti-IgG and anti-C3d are used to detect in-vivo or in-vitro coating of red cells
with antibody and/or complement. AHG reagent detects the presence of IgG immunoglobulin in the
plasma being tested following incubation with red cell reagents expressing the corresponding antigen. It
can be used to demonstrate human globulin molecules either bound to red cells or free in plasma.
IgM antibody activity is usually detected by employing a saline technique. Once the antibody is bound to
a red cell antigen, the larger size of the IgM molecule allows bridging between the antigens on adjacent
red cells in a saline suspension and produces agglutination.
IgG antibodies are smaller molecules and generally unable to cause direct agglutination in saline once
they are bound to a red cell antigen. Detection of IgG antibodies bound to a red cell may be achieved by
using bridging molecules, e.g. indirect antiglobulin test. Techniques to enhance reactions include
alteration of the red cell membrane e.g. use of proteases (enzyme test) and modification of the
suspension medium e.g. addition of macromolecules such as polyethylene glycol (PeG).
The direct antiglobulin test (DAT) is used to demonstrate the in-vivo coating of red cells with antibody
and/or complement components. If in-vivo coating has occurred, centrifugation of the red cells through a
LISS/Coombs ID-Card will allow the AHG reagent present in the ID-Card to agglutinate the red cells.
The indirect antiglobulin test (IAT) is used to demonstrate in-vitro reactions where free antibodies in
plasma coat but do not agglutinate red cells that express the corresponding antigen. To perform the IAT
test, red cells are incubated with plasma at 37ºC in a LISS/Coombs ID-Card, the antibody-antigen
reaction occurs during incubation. The antibody becomes attached to the red cells in a microtube above
a gel matrix containing AHG reagent. The gel card centrifugation method of separating the red cells
from liquid phase through the gel, eliminates the need to wash the red cells. With centrifugation non-
agglutinated cells form a pellet in the bottom of the microtube, whereas agglutinated cells are trapped in
the gel. The reaction strength is determined by the extent to which the agglutinated cells have been
impeded in their movement through the gel under centrifugation.
The Bio-Rad screening cells (ID-Diacell I-II-III) consist of 3 group O test cells expressing the following
antigens: C, c, D, E, e, M, N, S, s, P1, K, k, Fya, Fyb, Jka, Jkb, Lea, Leb, including R1R1 and R2R2 cells,
homozygous for Fya, Fyb, Jka, Jkb, M, and preferably for S, and s. When an antibody is detected by
routine IAT screening, identification of atypical antibodies (either autoantibodies or alloantibodies) is
achieved by testing the patient's plasma against the Bio-Rad ID-DiaPanel consisting of 11 group O adult
test cells which have been fully typed for the common inherited blood group antigens. If antibodies are
detected by IAT they may need a supplementary enzyme or IAT panel. Some laboratories may perform
an IAT tube test when further antibody identification is required. In the IAT tube test the patient's plasma
is incubated with the selected red cells (by saline IAT or with a low ionic additive) and the red cells are
subsequently washed and tested with AHG reagent for the presence of human globulin attached to the
red cells.
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Approver: Director Clinical Operations, Version Number: V1.0, Publication Date: 27/10/2021 This document is subject to
change and a printed copy may not be up to date. The current version is only available online in the
NSW Health Pathology Policy Library
w w w.p a t ho lo g y.h e a lt h .nsw.g o

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Detection of antibody activity by IAT is dependent upon the following:

The concentrations of antibody and antigen. The sensitivity of antibody detection tests is enhanced by
having a suitable cell to plasma ratio.
Length of incubation. The interaction between antigen and antibody is time dependent and tests should
be incubated for the required time period to allow sufficient antibody-antigen binding and permit
detection.
Temperature of incubation. The temperature of incubation determines the rate at which antigen-antibody
reactions occur. The rate of reaction of IgM antibodies is optimal at temperatures between 4ºC and 20ºC
and are detected by saline techniques which can be performed at 4ºC, 20ºC and 37ºC. IgG antibodies
prefer a reaction temperature of 37ºC and are mostly detectable by IAT only.
Ionic strength. A reduction in the ionic strength of the incubation mixture increases the rate at which
antigen and antibodies bind.
Enzyme techniques
Proteolytic enzymes cleave sialic acid molecules from polysaccharide chains reducing the net negative
charge at the red cell surface. The modification of the red cell membrane enables agglutination to take
place more readily, the reactivity of some antigen-antibody systems is enhanced (e.g. Rhesus, and Kidd)
and some antigenic configurations are destroyed or weakened (e.g. Duffy, Ss, M, N). Enzyme treatment
of red cells may be particularly useful to identify antibodies, where multiple antibodies are present. The
standard laboratory procedure is to use the Bio-Rad ID-DiaPanel-P (papanised) enzyme panel for
antibody investigation.
Addition of Macromolecules
The use of macromolecules such as polyethylene glycol (PeG), N-Hance or RAM-Rapid Antibody
Medium (RAM) as an additive in an IAT enhances the sensitivity of the antibody detection procedure. By
creating a low-ionic-strength test environment the rate at which antigen and antibody binds is increased
and the incubation time reduced.
1.2. Scope
This test method applies to all NSW Health Pathology scientific and technical staff involved in
pretransfusion laboratory practice.
1.3. Test Frequency and Expected Turnaround Time

Performed on request during laboratory hours.
Refer to local laboratory procedures for expected turnaround times.
1.4. Definitions
AHG: Anti-Human Globulin
IAT: Indirect Antiglobulin Test
CAT: Column Agglutination Technology
LISS: Low Ionic Strength Solution
PeG: Polyethylene Glycol
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RAM: Rapid Antibody Medium

EDTA: Ethylenediaminetetraacetic acid
LIS: Laboratory Information System
RCPA QAP: Royal College of Pathologists of Australasia Quality Assurance Program
ARCL: Australian Red Cross Lifeblood
Daratumumab: Anti-CD38 monoclonal antibody
Isatuximab: Anti-CD38 monoclonal antibody
Magrolimab: Anti-CD47 monoclonal antibody
DTT: Dithiothreitol. When DTT is used to treat red cells, it denatures cell surface CD38 by disrupting
disulphide bonds in the extracellular domain of CD38 and thereby prevents Daratumumab binding.
2. Method Specifications
2.1. Potential Sources of Assay Variability
 Bacterial or other contaminations of reagents can cause false positive or false negative results, look
for turbidity. Repeat test with new reagents.
 Fibrin in the plasma or red cell aggregates in the red cell suspension may trap non-agglutinated cells
at the top of the gel while most of the cells are on the bottom of the microtube after centrifugation.
Remove fibrin and repeat test.
 Elevated white cell counts may cause false positive reactions. Consult with senior laboratory staff.
 Too heavy or too light red cell suspensions may cause discrepant results.
 Correct pipetting technique to avoid contact of the pipette tip with the contents of the microtubes will
prevent carryover.
 Cord Blood collections - Wharton’s jelly is a gelatinous substance within the umbilical cord. It can
cause false agglutination. The control well should also be positive in this circumstance, rendering the
results invalid.
3. Pre-Analytical
3.1. Primary Specimen Type e.g. blood, urine, tissue
Venous blood collected aseptically.
3.2. Specimen containers including any additives, anticoagulants, buffers, acid

 An anticoagulated EDTA (pink or purple top) specimen tube centrifuged as per local policy prior to
testing.
 Other tube types that may be accepted for testing include citrate (blue top) clotted blood (red top) and
CPD-A centrifuged as per local policy prior to testing.
 Lithium heparin (green top) and serum gel tubes are not acceptable due to the potential for the gel to
absorb antibodies resulting in false negative results.
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Note: Throughout the document reference to plasma and serum is interchangeable.
3.3. Assay Validation and Verification

Validation and Verification documentation of the test method is reviewed and signed by the Supervising
Pathologist and retained in the Quality Management System. For further information refer to Verification
Procedure for the Bio-Rad IH 1000, IH 500 and Bio-Rad Saxo Analysers NSWHP_PR_046.
3.4. Sensitivity, Specificity of detection

The antibody screening and panel cells must be capable of detecting Anti-D at a concentration of
0.1IU/ml as per the ANZSBT guidelines. This is checked daily by running the Bio-Rad Basic QC.
3.5. Patient preparation e.g. fasting, time after last dose – if applicable
Not Applicable
3.6. Specimen Rejection Criteria

A specimen will be rejected for testing if:
 The specimen does not meet the criteria outlined in Minimum Patient Identification Requirements for
Pre-Transfusion Testing NSWHP_PD_009
 The specimen is insufficiently filled. Refer to local recollection procedures.
 The specimen is grossly haemolysed. Refer to local recollection procedures.
3.7. Specimen Transport Requirements – frozen, room temp, on ice, other

Specimens may be transported at room temperature or 2-8oC.
3.8. Specimen Stability, Storage and Retention

Specimens should be tested within 24 hours of collection wherever possible, and testing completed
within 48 hours when kept at room temperature (18-25ºC) and up to 7 days when stored between 2-8oC.
Specimens stored at 2-8oC are not used for serological testing beyond 7 days from the date of collection.
If specimens are required to be routinely tested after 7 days, they must be separated, and the plasma
stored frozen at -20 ºC for up to 3 months. Refer to local laboratory procedures for specimen retention
time.
4. Reagents, Supplies and Equipment

4.1. Required Reagents and Reagent Source
DiaClon ID-Card LISS/Coombs: Indirect and Direct Coombs Test (Product ID 50531)
DiaClon ID-Card Coombs Anti-IgG (Product ID 50540)
DiaClon ID-Card NaCl, Enzyme Test and Cold agglutinins (Product ID: 50520)
ID-Diacell (I-II-III) Test cell reagents for antibody screening (Product ID 45184)
ID-DiaPanel (Set of 11 vials) Test cell reagents for antibody identification- IAT (Product ID 45161)
ID-DiaPanel-P (Set of 11 vials) Test cell reagents for antibody identification- enzyme (ID 45171)
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ID-Diluent 2 modified LISS for red cell suspensions (Product ID 05761)

Abtectcell III, 0.8% Reagent red blood cells for antibody screening
Phenocell B and C, 0.8% Reagent red blood cells for antibody identification
Required Tube Reagents- Refer to Current Suppliers
Bio-Rad 3% Reagent red blood cells for antibody screening and antibody identification
Polyspecific and IgG AHG reagent
Antiglobulin Control Cells
LISS reagent refer to local procedure, N-Hance, RAM-Rapid Antibody Medium or PeG
0.9% Buffered Saline Solution pH 7.1
4.2. Reagent Preparation

Not Applicable
4.3. Reagent Stability and Storage

 ID-Diacell, ID-DiaPanel, ID-DiaPanel-P, Abtectcell III and Phenocell red cell reagents store at 2-8ºC
do not store near any heat, air conditioning sources or ventilation outlets. Red cell reagents must be
gently resuspended and at room temperature (18-25 °C) when used for manual testing. Reagents are
used until the expiry date as indicated by the manufacturer. Refrigerated reagents stored on board
the Bio-Rad analyser are stable for 7 days on the IH-500 and for 48 hours on the IH-1000.
 ID-Diluent 2 store at 2-8ºC do not store near any heat, air conditioning sources or ventilation outlets,
once opened expiry is 6 months.
 DiaClon ID-Cards store at 18-25ºC. Inspect cards before use ensuring that buffer covers the gel and
that there are no bubbles in the gel. DiaClon ID-Cards are used until the expiry date as indicated by
the manufacturer. ID-Cards stored on board the Bio-Rad analyser are valid for 21 days on the IH-500
and 7 days on the IH-1000.
 Tube reagents store at 2-8ºC do not store near any heat, air conditioning sources or ventilation
outlets. Reagents are used until the expiry date as indicated by the manufacturer.
 0.9% Buffered Saline Solution pH 7.1 store at room temperature.
4.4. Laboratory Equipment

Automated Bio-Rad CAT method
Testing is performed on the Bio-Rad IH-500 or Bio-Rad IH-1000, fully automated system for
immunohaematology diagnostics.
Manual Bio-Rad CAT Method
The Bio-Rad Saxo ID-Reader II is a semi-automated system for immunohaematology diagnostics and
relies heavily on the technician correctly following testing procedures. Manually pipetted gel cards are
incubated (if required), centrifuged and the reactions read by the integrated camera and the results
interpreted by the interpretation software.
 ID-Dispenser (for the Diluent)
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 Pipette and Pipette tips

 ID-Working table
 Test tubes
 ID-Incubator L
 ID-Centrifuge L (back up centrifuge)
Manual Tube Method
 Glass test tubes
 Plastic transfer pipettes
 Tube rack
 Tube centrifuge
 Cell washer – if available
4.5. Quality Control and QC Acceptance Criteria

Pre acceptance testing is performed on all new batches of ID-Diacell (I-II-III), ID-DiaPanel, ID-
DiaPanel-P, Abtectcell III, Phenocell red cell reagents and ID-Cards before being before being placed
into general use. Refer to Pre Acceptance Testing of Red Cell Reagents NSWHP_PR_051 and Pre-
Acceptance Testing, Transport and Storage of Bio-Rad ID-Cards and Solutions NSWHP_PR_050 for
further information.
Quality control for the Forward/Reverse Cards, Group Check Cards, LISS/Coombs ID-Cards, and ID-
Diacell (I-II-III) are performed daily using QC material. Bio-Rad IH-QC kit is the standard QC material.
QC results will be accepted for validation if the quality control is within range (strengths of reactions
consistent with the package insert). Any discrepancies must be investigated immediately, report QC out
of range to laboratory senior. Patient specimens will not be tested until QC results are acceptable. In the
case of QC failure that may have compromised patient results, all samples that have been tested since
the last valid QC will have to be repeated, and all blood products dispensed based on these results
reviewed immediately and acted upon.
4.6. Calibration Procedures

The calibration and preventative maintenance of the Bio-Rad Saxo ID-Reader II, ID-Incubator L and ID-
Centrifuge L is performed annually by qualified Bio-Rad engineers.
The calibration of the IH-500 and the IH-1000 is performed at 6 monthly preventative maintenance
services by qualified Bio-Rad engineers.
Pipette checks are performed every 6 months.
Centrifuge calibrations are performed annually.
4.7. Quality Assurance Programs

NSWHP Laboratories are enrolled in RCPA QAP programmes including General Transfusion
Phenotyping and Antibody Titre modules. Some laboratories are enrolled in additional QAP such as the
AIMS QAP program. NSWHP Transfusion Laboratories participate in an interlaboratory sample
exchange program for tests not covered by RCPA QAP. Refer to local Quality Assurance standard
operating procedures for further information. All staff must participate in the quality assurance program
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and undertake at least two exercises per year to ensure their gradings are correct and that all positive
tests are being detected.
5. Method Detail
5.1. Full Procedure
Automation is the preferred method. Results for patient testing performed on the Bio-Rad Saxo ID-
Reader, IH-500 and IH-1000 are stored in the IH-Com database along with the patient details, batch
numbers and expiry dates of reagents, cells, ID-Cards and Diluents. All staff are allocated a unique user
ID and password which allows traceability of the user actions performed at all stages of testing.
Test ID-Cards and Test Cell Reagents for Standard Method

Antibody screening Diaclon ID-Card LISS/Coombs (Anti-IgG + C3d)
ID-Diacell (I-II-III) Test cell reagents for antibody screening
Antibody Identification Diaclon ID-Card LISS/Coombs (Anti-IgG + C3d)
(IAT) ID-DiaPanel (11 vials) Test cell reagents for antibody identification
Antibody Identification DiaClon ID-Card NaCl Enzyme test and Cold Agglutinins
(Enzyme) ID-DiaPanel-P (11 vials) Test cell reagents for antibody identification
Passive Anti-D Panel ID DiaScreen Prophylax (6 vials) or ID-DiaPanel (11 vials) Test cell
(IAT) reagents for antibody identification. Refer to local laboratory
procedures.
Standard laboratory procedure is to perform an antibody identification panel if positive reactions are
detected in the antibody screen. A supplementary IAT or enzyme panel may also be indicated. If the
patient is post transfusion and the DAT is monospecific IgG positive an eluate may be required to identify
any antibody(s) attached to the patient’s cells which could be causing a delayed transfusion reaction.
Refer to Direct Antiglobulin Test and Elution Test Method NSWHP_PR_067.
RhD negative women known to have had RhD Immunoglobulin administered within the previous three
months may produce a weakly positive antibody screen. An IAT panel or a dedicated Rh negative screen
is performed to exclude other clinically significant alloantibodies.
Antibody identification must be resulted before blood is crossmatched. Refer to Order, Selection and
Release of Blood Products NSWHP_PD_022 and local laboratory procedures for further details.
CRITICAL: Every time you pick up a specimen to test, to aliquot, to load onto an instrument, to
enter a result, or for any other purpose in the Transfusion Laboratory, the identifiers of the patient
on the specimen label must be checked against the accession label, and/or the request form.
Refer to:
 Bio-Rad Saxo ID-Reader II NSWHP_PR_057, for instructions on how to assign a card, print a
barcode, centrifuge and read ID-Cards .
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 Bio-Rad IH-500 Immunohaematology Analyser NSWHP_PR_055 and Bio-Rad IH-1000

Immunohaematology Analyser NSWHP_PR_056 for information on processing specimens on the
analysers.
 Bio-Rad IH-Com Data Management Software NSWHP_PR_054 for instructions on how to review
results in IH-Com.
5.2. LISS/Coombs IAT by manual CAT method.
The manual CAT method is performed using the Saxo ID-Reader II, refer to Bio-Rad Saxo ID-Reader II
NSWHP_PR_057 for instructions on how to assign ID-Cards and print a barcode label.
5.2.1. For each test label three microtubes of a DiaClon LISS/Coombs ID-Card with a barcode with
patient and reagent details.
5.2.2. Remove the aluminium foil from as many microtubes as required holding the ID-Card in the upright
position.
5.2.3. Pipette 50 µl of 0.8% red cell reagent to the appropriate microtubes.
a. For an antibody screening test
Pipette 50 µl of the ID-Diacell (I, II, III) to the microtubes marked with the corresponding test
cell 1 to 3.
b. For an antibody identification test
Prepare a 0.8% cell suspension of the patient's red cells for the autocontrol. In a test tube
labelled with patient identifiers pipette 1.0 mL of Diluent 2 and 10 µl of patient packed red
cells (centrifuged specimen) and mix gently.
Pipette 50 µl of the ID-DiaPanel test cells to the appropriate microtubes marked with the
corresponding test cell 1 to 11.
Pipette 50 µl of 0.8% patient red cell suspension to microtube 12 (autocontrol).
5.2.4. Pipette 25 µl of the patient plasma to each microtube.
5.2.5. Incubate the ID-Card(s) for 15 minutes at 37ºC in the ID-Incubator L.
5.2.6. Centrifuge and read the ID-Card(s) using the Saxo-ID-Reader II.
5.2.7. Record the results on the correct panel sheet ensure the results are labelled IAT test.
5.2.8. Refer to 7.1 Result Interpretation.
The IAT may be performed by the same method using a Coombs Anti-IgG ID-Card to remove
interference from complement components which may be non-specifically bound to the red cells.
An autocontrol (patient cells vs patient plasma) is set up with the panel to exclude autoantibodies.
If the autocontrol is positive perform and report the DAT. Refer to Direct Antiglobulin Test and
Elution Test Method NSWHP_PR_067.
5.2.9. Refer to LIS standard operating procedures for result entry details.
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Figure 1: Antibody screening using ID-DiaCell I-II-III
Figure 2: Antibody identification using ID-DiaPanel and auto control.
5.3. LISS/Coombs IAT by automated CAT method

The test is performed on the Bio-Rad IH-500 or Bio-Rad IH-1000 using the same volumes and same
dilutions as outlined in 5.2 LISS/Coombs IAT manual CAT method.
Refer to 5.6 Ordering additional tests in IH-Com for instructions on how to request a test not ordered in
the LIS.
5.4. Enzyme Test by manual CAT method

Bio-Rad ID-DiaPanel-P papain method is the standard laboratory procedure.
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5.4.1. Label the appropriate number of microtubes of the DiaClon NaCl Enzyme test ID-Card with a
barcode with patient and reagent details.
5.4.2. Remove the aluminium foil from as many microtubes as required holding the ID-Card in the upright
position.
5.4.3. Pipette 50 µl of the ID-DiaPanel-P test cells to the appropriate microtubes marked with the
corresponding test cell 1 to 11.
5.4.4. Pipette 25 µl of the patient plasma to each microtube.
5.4.5. Incubate the ID-Card(s) for 15 minutes at 37ºC in the ID-Incubator L.
5.4.8. Record the results on the correct panel sheet and ensure the results are labelled as enzyme test.
Figure 3: Antibody identification using ID-DiaPanel-P and autocontrol (not performed routinely)
5.5. Enzyme Test by automated CAT method

The test is performed on the Bio-Rad IH-500 or Bio-Rad IH-1000 using the same volumes and same
dilutions as outlined in 5.4 Enzyme Test by manual CAT method.
Refer to 5.6 Ordering additional tests in IH-Com for instructions on how to request a test not ordered in
the LIS.
5.6. Ordering Additional Tests in IH-Com.

5.6.1. Ordering Additional Tests for Patients on the Worklist:
1. At the Main (Analyser) screen, select ‘Data Management’.
2. Select the ‘’Work List tab.
3. Select the required patient (scroll down if necessary).
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4. Right Click and select ‘Request More’.

 For antibody screening select (check the box) Ab. Screening I-II-III (IAT) 5053- 3 cell screen
from the list on the right hand side of the screen.
 For an IAT panel select (check the box) Identification: 11 test cells IAT + AC (5053) from the
list on the right hand side of the screen.
If required load the ID-DiaPanel reagent red cells (or for a secondary IAT panel on the IH-1000
load the 0.8% Phenocell panel) onto the analyser using the reagent tray.
 For an Enzyme panel select (check the box) Identification: 11 cells test P Enzyme
(5052/5052).
If required load NaCl, Enzyme and Cold Agglutinins ID-Cards to the analyser card drawer and
load the ID-DiaPanel-P reagent red cells onto the analyser using the reagent tray.
5. Select ‘Save’.
6. For the IH-500 and IH-1000, put the patient sample into the sample rack and load onto the
analyser.
5.6.2. Ordering Additional Tests for Patients not on the Worklist:
1. At the Main (Analyser) screen, select ‘Data Management’.
2. Select the ‘Work List’ tab.
3. Right Click and select ‘Quick Request’.
4. Select the test from the dropdown list.
 For antibody screening select (check the box) Ab. Screening I-II-III (IAT) 5053- 3 cell screen
from the list on the right hand side of the screen.
 For an IAT panel select (check the box) Identification: 11 test cells IAT + AC (5053) from the
list on the right hand side of the screen.
If required load the ID-DiaPanel reagent red cells (or for a secondary panel on the IH-1000 load
the 0.8% Phenocell) onto the analyser using the reagent tray.
 For an Enzyme panel select (check the box) Identification: 11 cells test P Enzyme
(5052/5052).
If required load NaCl, Enzyme and Cold Agglutinins ID-Cards to the analyser card drawer and
load the ID-DiaPanel-P reagent red cells onto the analyser using the reagent tray.
5. Scan the sample barcode, IH-Com may identify the patient, A window box will open with ‘The
sample number is already in use for another patient: Surname,Firstname,Dob (Sex)[MRN}’
select ‘Yes’ if this is the correct patient.
6. Click ‘Order Now’ and ‘Close’.
7. For the IH-500 and IH-1000, put the patient sample in the sample rack and load onto the analyser.
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6. Result Out-Of-Range / Reflex Testing / Supplementary Testing

6.1. 4ºC Saline IAT by CAT method
Saline screening is a supplementary technique for patients who already have an antibody detected by
routine methods and can be performed at 4°C, 18-25°C (room temperature), or 37°C. Cold antibodies
that are not active at 37°C are unlikely to be clinically significant. It is used to help identify and enhance
cold antibodies that may be persisting through to IAT such as anti-M, -N, -P1, -Lea, -Leb and -I. Saline
techniques are also used to define haemolytic anaemia associated with cold antibodies.
6.1.1. Label a DiaClon NaCl ID-Card(s) with patient and reagent (and temperature details). Prior to use,
the NaCl card is to be kept at 2-8oC for a minimum of 2 hours. It is important to use refrigerated
red cell reagents and patient samples.
6.1.2. If an autocontrol is required prepare a 0.8% suspension of patient cells in ID-Diluent 2. In a test
tube labelled with patient identifiers pipette 1 mL of Diluent 2 and add 10µl of patient red cells, mix
gently and place the tube in the refrigerator for 30 minutes.
6.1.3. Remove the aluminium foil from as many microtubes of the ID-Card as required, holding the ID-
Card in the upright position.
6.1.4. Pipette 50 µl of 0.8% red cell reagent to the appropriate microtubes.
a. For Antibody Screening
cell 1 to 3.
b. For Antibody Identification
Pipette 50 µl of the ID-DiaPanel test cells to the microtubes marked with the corresponding test
cell 1 to 11.
Pipette 50 µl 0.8% patient red cell suspension to microtube 12 (autocontrol).
6.1.5. Add 25 µL of patient plasma to each well.
6.1.6. Incubate the ID-Card(s) for 30 minutes at 2-8oC.
6.1.8. Record the results on the correct panel sheet and ensure the results are labelled as 4°C saline
test.
6.1.9. See 7.1 for Result Interpretation.
6.2. 37ºC Pre-Warmed LISS/Coombs IAT by CAT method

If a patient has cold agglutinins repeat the IAT test at 37oC with pre-warmed cards and reagents.
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6.2.1. For prewarmed testing at 37oC place a DiaClon LISS/Coombs ID-Card(s) labelled with patient and
reagent details into the 37oC incubator for 30 minutes.
6.2.2. If an autocontrol is required prepare a 0.8% suspension of patient cells in ID-Diluent 2. In a test
tube labelled with patient identifiers pipette 1 mL of Diluent 2 and add 10 µl of patient red cells,
mix gently and place the tube in the 37oC incubator for 30 minutes.
6.2.3. Separate an aliquot of patient plasma into a labelled tube and place in the 37oC incubator for 30
minutes.
6.2.4. Pipette 60 µl of red cell reagents into separate labelled tubes and place in the 37oC incubator for
30 minutes . Do not place whole bottles in the incubator .
6.2.5. Remove the aluminium foil from required microtubes of the ID-Card, holding the ID-Card in the
upright position and place in the 37oC incubator.
6.2.6. Keep the pre-warmed LISS/Coombs cards in the incubator while pipetting 0.8% red cell reagents
to the appropriate microtube.
cell 1 to 3.
Pipette 50 µl of the ID-DiaPanel test cells to the microtubes marked with the corresponding
test cell 1 to 11.
Pipette 50 µl of pre-warmed 0.8% patient red cell suspension to microtube 12 (autocontrol).
6.2.7. Add 25 µl of patient pre-warmed plasma to each well.
6.2.8. Incubate the ID-Card(s) for 15 minutes at 37oC in the ID-Incubator L.
6.2.10. Record the results on the correct panel sheet and ensure the results are labelled as 37oC IAT
test performed with prewarmed card and reagents.
If the patient has strong cold agglutinins the test may need to be repeated with a new patient
sample collected and kept at 37oC
6.3. Saline IAT by tube method

6.3.1. For each test label glass test tubes with two patient identifiers and the appropriate test cells.
 For each test label three tubes 1 to 3 with two patient identifiers.
 Add 2 drops of patient's plasma to each tube using a transfer pipette.
 Add one drop of the 3% screening cells to the respective tube and mix well.
Page 13 of 25

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NSWHP_PR_062

If an autocontrol is required prepare a 3% suspension of the patient cells in buffered saline.
 Label a test tube with patient identifiers and add one drop of centrifuged patient red cells
with a plastic transfer pipette. Wash cells once in saline, to wash add buffered saline
(3/4 full) to the labelled tube and centrifuge tube for 1 minute at high speed (900 rcf).
Decant the supernatant off and discard. Use the remaining patient red cells for a cell
suspension of washed patient cells.
 Label a clean test tube with patient identifiers. To make a 3% red cell suspension,
pipette 500 µl buffered saline as diluent and add 15 µl of washed patient red cells and
mix gently.
For each test label 12 tubes 1 to 11 and AC (autocontrol) with two patient identifiers.
Add 2 drops of patient's plasma to each tube using a transfer pipette.
Add one drop of the 3% panel cells to respective tube labelled 1 to 11 and mix well.
Add one drop of 3% patient red cells to tube 12 as labelled and mix well.
6.3.2. Incubate all the tubes for 30 minutes at 37oC.
6.3.3. After 30 minutes centrifuge test tubes at high speed (900 rcf) for 15 seconds.
6.3.4. Inspect for haemolysis and resuspend the red blood cells by gently shaking and examine for
agglutination then record results.
6.3.5. Wash the cells in the tubes with four changes of saline. To manually wash cells, dispense 4 mL of
buffered saline into each tube and mix well. Centrifuge the tubes for 60 seconds high speed (900
rcf). Decant the supernatant off and discard after each wash, ensuring the cells are resuspended
between washes. Repeat three times. Or if available, transfer the tubes to the automatic cell
washers and set for four washes with buffered saline. Do not remove tubes from 37oC incubation
until cell washers are available.
6.3.6. To the ‘dry button’ of red cells after the final wash add 2 drops of polyspecific AHG reagent and
mix thoroughly.
IgG AHG reagent may be used instead of polyspecific AHG reagent to avoid reactivity with
complement components in patients with autoantibodies.
6.3.7. Centrifuge the tubes (refer to manufacturers package inserts for centrifuge speed and time).
6.3.8. Read tubes macroscopically by gentle agitation and record results.
6.3.9. Add one drop of AHG control cells to all tubes giving a negative result and mix well.
6.3.10. Centrifuge the tubes at high speed (900 rcf) for 15 seconds.
6.3.11. Read tubes macroscopically by gentle agitation The antiglobulin control cells must be
agglutinated in each tube for negative results. If the AHG control cells are not agglutinated the test
is not valid and must be repeated. The expected reaction grade is ≥ 2+.
6.3.12. Refer to 7.1.2 Result Interpretation.
Page 14 of 25

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NSWHP_PR_062
6.4. LISS/Coombs IAT by tube method

6.4.1. Perform step 6.3.1 of 6.3. Saline IAT by tube method.
6.4.2. Add 2 drops of LISS (CSL RAM, Gamma N-Hance or PeG) to each tube and mix well.
6.4.3. Incubate all the tubes for 10 minutes at 37oC. Incubation time must be at least 10 minutes and not
more than 30 minutes.
6.4.4. Centrifuge tubes (refer to manufacturers package inserts for centrifuge speed and time). Note: if
PeG additive has been used tubes must not be centrifuged prior to washing.
6.4.5. Inspect for haemolysis and resuspend the red blood cells by gently shaking and examine for
agglutination then record results.
6.4.6. Wash the cells in the tubes with four changes of saline. To manually wash cells, dispense 4 mL of
buffered saline into each tube and mix well. Centrifuge the tubes for 60 seconds at high speed
(900 rcf). Decant the supernatant off and discard after each wash, ensuring the cells are
resuspended between washes. Repeat three times. Or if available, transfer the tubes to the
automatic cell washers and set for four washes with buffered saline. Do not remove tubes from
37oC incubation until cell washers are available.
6.4.7. To the ‘dry button’ of red cells after the final wash add 2 drops of polyspecific AHG reagent (IgG
AHG reagent if using PeG) and mix thoroughly.
6.4.8. Centrifuge the tubes (refer to manufacturers package inserts for centrifuge speed and time).
6.4.9. Read tubes macroscopically by gentle agitation record results.
6.4.10. Add one drop of AHG control cells to all tubes giving a negative result and mix well.
6.4.11. Centrifuge the tubes at high speed (900 rcf) for 15 seconds.
6.4.12. Read tubes macroscopically by gentle agitation. The AHG control cells must be agglutinated in
each tube for negative results. If the AHG control cells are not agglutinated the test is not valid
and must be repeated. The expected reaction grade is ≥ 2+.
6.4.13. Refer to 7.1.2 Result Interpretation.
6.5. Calculations
Not Applicable.
7. Reporting Results
One or more positive reactions indicate the presence of irregular antibodies, enter the reactions obtained
on the respective panel sheet, and the method by which testing was performed (e.g. IAT, enzyme).
Verify that the lot number of the test cell reagents correspond to the lot number indicated on the panel
sheet. Refer to local LIS standard operating procedures for result entry.
7.1. Result Interpretation and choice of Coded Comments

Positive: Agglutinated cells forming a red line on the surface of the gel or dispersed in the gel.
Negative: Compact button of cells on the bottom of the microtube.
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7.1.1. Reaction Grading for Bio-Rad CAT Testing
7.1.2. Reaction Grading for Tube Testing
7.2. Antibody Identification

The antigen profile of the cells included on a panel cover only a proportion of the total antigens
represented on each red cell; hence the reporting of antibodies is done in terms of the most probable
antibody present.
If positive and negative reactions are observed with different test cells, antibody identification is
simplified by considering the negative reactions first and excluding antibodies. The specificity of an
antibody can be normally be assigned when it is reactive with at least two red cells carrying the
corresponding antigen and non reactive with at least two red cells lacking the antigen. The
presence of Anti-Jka, Anti-Jkb, Anti-Fya, Anti-Fyb, Anti-S, Anti-s, Anti-M and Anti-N should be excluded
using red cells with homozygous expression of the corresponding antigens.
If all antigens are excluded this may indicate that a single antibody is present reacting only against
cells having strong antigen expression, such as cells homozygous for an antigen. Re-examine the
panel giving attention to those antigens that are known to show dosage e.g. c, E, Duffy, Kidd and M.
Some other antigens may vary widely in antigen strength which is unrelated to the zygosity of the gene
producing the antigen, e.g. P1, Lewis.
If the reactions do not indicate a single common antibody it is best to assume that the reactions are due
to a mixture of common antibodies before considering less common antibodies.
Clear identification of antibodies of clinical significance is not always possible. A second panel may be
required to help identification the antibody. If Cw, Kpa, Lea, Leb, Lua antigens have not been excluded a
second panel may not be required, these antibodies are not considered clinically significant and only
require an IAT compatible crossmatch. Refer to local laboratory procedure.
Once the specificity of the antibody/ies has been established it is necessary to ensure that the patient’s
red cells lack the corresponding antigen. The patients red cells usually lack the antigen against which
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NSWHP_PR_062
the antibody is directed unless the antibody is an autoantibody. Patient antigen typing should be
confirmed using Bio-Rad antigen phenotyping ID-Cards (or commercial antisera) if the patient has not
been transfused in the last 3 months.
Factors to consider when specificity cannot be determined;
 Phases of reactivity, saline, enzyme, IAT
 Dosage effects with homozygous as compared to heterozygous cells and complement dependency
 Variable grades of reactivity and multiple antibodies
 Presence of haemolysis
 Reactivity with autologous controls
 Reactivity with ABO compatible cells or cord blood cells
 Reactivity with reagent additives, drug dependency or pH dependency
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Table 1: Antibody Identification Procedures.
For Positive Reactions With all ID-DiaPanel Test Cells
If all panel cells are positive as well as the autocontrol, showing the same strength this may
be an autoantibody. Patients with autoantibodies must have a Rh and K phenotype
performed. Extended phenotyping (pretransfusion) can assist with antibody identification
and/or selection of phenotype matched red cells (Rh, K/k, Fya/b, Jka/b, MS/s) for transfusion.
Patient’s may be referred for a molecular genotype if a serological phenotype cannot be
obtained due to, recent transfusion, positive DAT, ambiguous results or confirmation of Fy(a-b-)
GATA mutation. Autoantibodies can mask significant alloantibodies. Refer to local procedures
and Autoantibody and Autoadsorption Test Methods NSWHP_PR_064 for further
investigation procedures.
Negative auto control Nonspecific reactions may indicate the presence of an

alloantibody directed against a high frequency antigen. High
titre low avidity antibodies (HTLA) such as Chido /Rodgers
react with most cells on the panel. Refer to ARCL for
identification.
Positive auto control Perform and report DAT and monospecific DAT. Refer to Direct
Antiglobulin Test and Elution Method NSWHP_PR_067.
One or more test cells May indicate an underlying alloantibody and further
showing stronger reactions investigation may be required. Refer to local procedure.
than the positive auto
control
For Antenatal Patients
If the antibody detected is Anti-D check the patient’s history for evidence of RhD
Immunoglobulin administration and record the date of administration. If the antibody screen is
≥ 2+ then the patient will have to be further investigated as the anti-D may be immune rather
than passively acquired. It is extremely important that an immune anti-D is not interpreted as a
passive anti-D.
If reactions are suggestive of an immune response an anti-D titre MUST be performed if:
 RhD Immunoglobulin was administered in the previous 8-12 weeks and the reaction
strength is >2.
 RhD Immunoglobulin was administered greater than 8-12 weeks before sample collection
or there is no history of RhD Immunoglobulin being administered.
If a clinically significant antibody is detected by the identification panel an antibody titre should
be performed. Refer to Antibody Titration Test Method NSWHP_PR_063.
If Anti-D and/or anti-c is detected the sample is sent to ARCL for antibody quantitation. The
patient should be referred to a specialist fetal medicine unit for assessment and monitoring if
they have an anti-D level ≥ 4 IU/mL or titre ≥ 32 or a significant rise in titre.
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Monoclonal Immunotherapies Affecting Pretransfusion Testing
A number of immunotherapies may cause panagglutinins which can interfere with

pretransfusion testing and significantly delay the provision of blood. Agglutination to Anti-
CD38 and Anti-CD47 may occur in all media (e.g. Saline, LISS, PeG) and by both CAT and
tube IAT methods.
Daratumumab is a monoclonal immunological treatment for relapsed multiple myeloma, which
recognizes CD38 on myeloma cells. CD38 is strongly expressed on myeloma cells but is also
weakly expressed on red blood cells and therefore causes red cell panagglutinins reacting 1+
to 2+ with all red cells by IAT. The red cell panagglutinins are resistant to autoadsorption. The
autocontrol may be positive and interfere with serological phenotyping of the patient by the
IAT method. DTT treatment of reagent red cells removes CD38 (as well as all the Kell system
and other clinically significant minor antigens such as Lutheran) and allows underlying
antibodies to be detected. Anti-CD38 does not interfere with ABO-RhD typing. Ficin treated
red cells are non-reactive with Daratumumab patients. Daratumumab-medicated positive IAT
may persist for up to 6 months after the last Daratumumab infusion.
Anti-CD47 (Hu5F9-G4) a glycoprotein expressed on all cells, binds to signal-regulatory
protein α on macrophages and regulates phagocytosis. Blocking CD47 is thought to enhance
phagocytosis and promote antitumor responses resulting in a panagglutinin reacting 3+ to 4+
with all red cells by all methods including IAT and immediate spin tests (e.g. reverse plasma
blood grouping). RhD negative red cells react stronger than RhD positive red cells by IAT.
Initially the panagglutinin reactions are weak and the discrepancy can be resolved by saline
IgG AHG tube method. If the ABO group cannot be concluded, group O red cells may be
required for transfusion. In patients who have a positive DAT, adsorption studies can allow
satisfactory antibody identification in many cases.
Protocol:
a. The treating Clinician or Trial Coordinator should notify the laboratory if a patient is on or
starting these immunotherapies.
b. The laboratory should record an alert in the LIS.
c. Determine the patients pretransfusion extended phenotype or genotype and provide
matched red cell transfusions for Rh and K, and where possible also for Fya, Fyb, Jka, Jkb,
S, s negative antigens to avoid alloantibody production. Where pretransfusion extended
phenotyping is not available and the patients has been transfused with non phenotype
matched blood in the previous three months and has uniform panagglutination indicative
of monoclonal immunotherapy interference the sample should be referred to ARCL.
d. Panel results must be checked closely for any variable reactivity which may indicate the
presence of alloantibodies. Any significant variation in reaction strength or haemolysis in
the panel results, which may indicate the presence of an alloantibody must be reported
immediately to the laboratory senior, e.g. any reaction strength > 2+ or variable reaction
strengths for patients on Anti-CD38 must be reported to the laboratory senior. Patients
with evidence of unexplained haemolysis, significant variation in reaction strengths of the
pan-agglutinating antibody, or a significant change in strength to the previous results,
should be referred to ARCL.
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Monoclonal Immunotherapies Affecting Pretransfusion Testing
These results may be suggestive of a developing alloantibody may require antibody screening
against a DTT treated panel for investigation
Crossmatching:
When the panagglutination is present serologically crossmatched blood will be incompatible,
and resistant to autoadsorption for Anti-CD38. All crossmatches must be reported using
untreated plasma and an autocontrol must be performed in parallel. Red cell units will
therefore be issued as incompatible and must be approved by a Haematologist or
Haematology registrar. The Haematologist or Haematology Registrar may authorise the
continuous issuing of incompatible blood without prior approval on a patient by patient basis
Page 20 of 25

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Table 2: Antibody Detection Method

Antibody Detection Method Additional Information
Anti-D IAT, enhanced by enzyme
Anti-C IAT, enhanced by enzyme Commonly found with Anti-D.
Anti-C+D IAT, enhanced by enzyme If pregnant Consider Anti-G (Refer to ARCL)
Anti-c IAT, enhanced by enzyme Commonly found with Anti-E.
Anti-E IAT, enhanced by enzyme
Anti-e IAT, enhanced by enzyme Common autoantibody
Auto-anti-e. Transfuse blood matching patient
Rh and K phenotype.
Alloantibody: Transfuse with e neg blood.
Anti-K IAT +/- weak reactions with enzyme
Anti-Fya IAT, non-reactive by enzyme
Anti-Fyb IAT, non-reactive by enzyme
Anti-S IAT, non-reactive by enzyme
Anti-s IAT
Anti-M Saline, sometimes IAT, Commonly reacts stronger at 4oC.
non-reactive by enzyme Can interfere with a patient’s reverse group.
Anti-N Saline, sometimes IAT,
non-reactive by enzyme
Anti-Kpa IAT Low incidence antigen – 99% of donors are
Kpa Negative.
Anti-Jka IAT enhanced by enzyme and
PeG
Anti-Jkb IAT enhanced by enzyme and Very Rare.
PeG
Anti-Cw IAT, enhanced by enzyme Low incidence antigen – 99% of donors are
Cw negative.
Anti-k IAT Very Rare. Transfuse with k negative blood.
Contact senior staff.
Page 21 of 25

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7.3. Critical Alert Values or Notifications

Refer to local standard operating procedures.
Table 3: The clinical significance of red cell alloantibodies and selecting blood for
transfusion6,7.
Antibody Specificity Clinically Significant Selection of Units
Anti-A1 Rarely IAT crossmatch compatible at 37oC
Anti-HI (A1 and A1B individuals) Rarely IAT crossmatch compatible at 37oC
Anti-M (active at 37oC) Rarely Antigen Negative
Anti-N (active at 37oC) Rarely IAT crossmatch compatible at 37oC
Anti-S, -s, -U Yes Antigen Negative
Anti-P1 (active at 37oC) Rarely IAT crossmatch compatible at 37oC
Anti-D, -C, -c, -E, -e Yes Antigen Negative
Anti-Cw Rarely IAT crossmatch compatible at 37oC
Anti-Lua Rarely IAT crossmatch compatible at 37oC
Anti-Lub Yes Antigen Negative (contact ARCL)
Anti-K, -k Yes Antigen Negative
Anti-Kpa Rarely IAT crossmatch compatible at 37oC
Anti-Lea, -Leb, -Lea+b Rarely IAT crossmatch compatible at 37oC
Anti-Fya, -Fyb Yes Antigen Negative
Anti-Jka, -Jkb Yes Antigen Negative
Anti-Coa Yes Antigen Negative
Anti-Cob Sometimes IAT crossmatch compatible at 37oC
Anti-Wra Rarely IAT crossmatch compatible at 37oC
7.4. In-House IVD class 1-3 or other Disclaimer

Not Applicable
7.5. Record Retention

Refer to NPAAC Retention of laboratory records8.
8. Roles and Responsibilities

1. The Supervising Pathologist discharges the supervision responsibilities as set out in the NPAAC
Requirements and Supervision Requirements, including documented review of audits, new tests
and equipment, external QAP, internal Quality Control and Quality Assurance10.
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NSWHP_PR_062
2. Laboratory managers are responsible for ensuring staff are trained in the use of this test method
and are deemed competent. Managers are also responsible for ensuring that records are retained
according to NPAAC requirements8.
3. Technical and Scientific staff are responsible for performing the test and reporting the results as
documented in this procedure.
9. Legal and Procedure Framework

1.
Saxo ID Reader II User Manual, DiaMed GmbH Version 2- 08/2016, SAP H007416
2.
IH-500 User Manual, DiaMed GmbH Version 2.2- 11/2018, DOC H009225
3.
IH-1000 User Manual, DiaMed GmbH Version 1.5- 07/2017, SAP H009142
4.
IH-Com user Manual, DiaMed GmbH Version 2-04/2019 DOC H009146
5.
NPAAC Requirements for Medical Pathology Services, Third Edition 2018 (Tier 2)
https://www1.health.gov.au/internet/main/publishing.nsf/Content/health-npaac-docs-
medpathserv-2018
6. NPAAC Requirements for Transfusion Laboratory Practice, Fourth Edition 2019 (Tier 4)
http://www.health.gov.au/internet/main/publishing.nsf/Content/npaac-pub-transfusion
7. ANZSBT Guidelines for Transfusion and Immunohaematology Laboratory Practice, Australian
and New Zealand Society of Blood Transfusion Inc 1st Edition, revised January 2020
https://anzsbt.org.au/guidelines-standards/anzsbt-guidelines/
8. NPAAC, Requirements for the Retention of Laboratory Records and Diagnostic Material, Eighth
Edition 2021 (Tier 3) https://www1.health.gov.au/internet/main/publishing.nsf/Content/health-npaac-
docs-RetLabRecDI-2018
9. NPAAC, Requirements for Supervision in the Clinical Governance of Medical Pathology
Laboratories, Sixth Edition 2021
https://www1.health.gov.au/internet/main/publishing.nsf/Content/health-npaac-docs-supervision.htm
10. NSW Health Pathology Supervision Policy NSWHP_PD_019
11. Reid M, Lomas-Francis, C. The Blood Group Antigen Facts Book, Elsevier, 3rd Edition, 2012
12. Harmening, D. Modern Blood Banking and Transfusion Practices 7th Edition, F. A. Davis, 2018
13. American Association of Blood Banks, Bethesda MD. AABB Technical Manual 20TH edition, 2020
Manufacturer’s Package Inserts
ID-Diacell I-II-III 0.8% and 3% Test cell reagents for antibody screening
ID-DiaPanel 0.8%, 3% and ID-DiaPanel-P 0.8% Test cell reagents for antibody identification
Abtectcell III 0.8% Reagent red blood cells for antibody screen
Phenocell B and C 0.8% Reagent red blood cells for antibody Identification
DiaClon ID-Card LISS/Coombs (and Coombs Anti-IgG): Indirect and Direct Coombs Test
DiaClon ID-Card NaCl, Enzyme Test and Cold agglutinins
ID-Diluent 2 modified LISS for red cell suspensions
Refer to Current Manufacturer’s Package Inserts.
Polyspecific and IgG AHG reagent
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Antiglobulin Control Cells

PeG, N-Hance or RAM-Rapid Antibody Medium (RAM)
9.1. Related Procedure Document Suite

Minimum Patient Identification Requirements for Pre-Transfusion Testing NSWHP_PD_009
Specimen Storage and Validity Criteria NSWHP_PR_023
Ordering Reagent Red Cells for Transfusion Testing NSWHP_PR_024
Order, Selection and Release of Blood Products NSWHP_PD_022
Quality Control for the Bio-Rad Immunohaematology Analysers NSWHP_PR_053
Pre Acceptance Testing of Red Cell Reagents NSWHP_PR_051
Pre Acceptance Testing, Transport and Storage of Bio-Rad ID-Cards and Solutions NSWHP_PR_050
Verification Procedure for the Bio-Rad IH 1000, IH 500 and Bio-Rad Saxo Analysers NSWHP_PR_046
Bio-Rad IH-Com Data Management Software NSWHP_PR_054
Bio-Rad Saxo ID-Reader II NSWHP_PR_057
Bio-Rad IH-500 Immunohaematology Analyser NSWHP_PR_055
Bio-Rad IH-1000 Immunohaematology Analyser NSWHP_PR_056
Direct Antiglobulin Test and Elution Method NSWHP_PR_067
Antibody Titration Test Method NSWHP_PR_063
Autoantibody and Autoadsorption Test Methods NSWHP_PR_064
Panel sheets for identification panels in use
10. Review
This procedure will be reviewed by 14/10/2023.
11. Risk
Risk Statement If the antibody screening and antibody identification of a pre-
transfusion specimen is not determined accurately, the
consequences could result in major harm to the patient.
Risk Category Clinical Care and Patient Safety,
11.1. Key WHS Information and Controls

Key WHS Factors and Risks WHS Controls
All specimens and reagents should Wear PPE while handling samples or if a spill occurs. Refer to
be treated as potentially infectious spill clean-up instructions and product safety data sheets for
further details.
Page 24 of 25

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12. Further Information

For further information, please contact:
Procedure Contact Officer Position: Chair, Transfusion Clinical Stream

Name: Associate Professor Mark Dean
Telephone: (02) 4320 3894
Email: Mark.Dean@health.nsw.gov.au
13. Version History

The approval and amendment history for this document must be listed in the following table.
Version No Effective Approved Approval Procedure Risk Rating Sections

Date By Date Author Modified
V1.0 27/10/2021 Director 15/10/2021 Associate High New Test
Clinical Professor Procedure
Operations Mark Dean
Page 25 of 25

NSWHP - PR - 062 - Antibody Screening and Identification Test Methods

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Antibody Screening and Identification Test

1.1. Principle of Assay

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Detection of antibody activity by IAT is dependent upon the following:

1.3. Test Frequency and Expected Turnaround Time

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RAM: Rapid Antibody Medium

3.2. Specimen containers including any additives, anticoagulants, buffers, acid

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Note: Throughout the document reference to plasma and serum is interchangeable.

3.3. Assay Validation and Verification

3.4. Sensitivity, Specificity of detection

3.6. Specimen Rejection Criteria

3.7. Specimen Transport Requirements – frozen, room temp, on ice, other

3.8. Specimen Stability, Storage and Retention

4. Reagents, Supplies and Equipment

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ID-Diluent 2 modified LISS for red cell suspensions (Product ID 05761)

4.2. Reagent Preparation

4.3. Reagent Stability and Storage

4.4. Laboratory Equipment

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 Pipette and Pipette tips

4.5. Quality Control and QC Acceptance Criteria

4.6. Calibration Procedures

4.7. Quality Assurance Programs

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Test ID-Cards and Test Cell Reagents for Standard Method

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 Bio-Rad IH-500 Immunohaematology Analyser NSWHP_PR_055 and Bio-Rad IH-1000

5.2. LISS/Coombs IAT by manual CAT method.

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Figure 1: Antibody screening using ID-DiaCell I-II-III

Figure 2: Antibody identification using ID-DiaPanel and auto control.

5.3. LISS/Coombs IAT by automated CAT method

5.4. Enzyme Test by manual CAT method

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5.5. Enzyme Test by automated CAT method

5.6. Ordering Additional Tests in IH-Com.

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4. Right Click and select ‘Request More’.

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6. Result Out-Of-Range / Reflex Testing / Supplementary Testing

6.2. 37ºC Pre-Warmed LISS/Coombs IAT by CAT method

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6.3. Saline IAT by tube method

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b. For Antibody Identification

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6.4. LISS/Coombs IAT by tube method

7.1. Result Interpretation and choice of Coded Comments

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7.1.1. Reaction Grading for Bio-Rad CAT Testing

7.1.2. Reaction Grading for Tube Testing

7.2. Antibody Identification

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Table 1: Antibody Identification Procedures.

For Positive Reactions With all ID-DiaPanel Test Cells

Negative auto control Nonspecific reactions may indicate the presence of an

For Antenatal Patients

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Monoclonal Immunotherapies Affecting Pretransfusion Testing